Keratinocyte transglutaminase (TGK) is a membrane protein responsible for cross-linked envelope formation during terminal differentiation in epidermal cells. In cultured keratinocytes, involucrin is its major substrate. Ordinarily neither protein is present in basal cells; expression occurs in maturing cells committed to terminal differentiation. The first part of the proposal exploits this lab's recent success in identifying response elements in the proximal promoter that are important for expression of the TGK gene. The DNA sequence requirements for protein binding to these elements will be determined by saturation binding mobility shift assay (to obtain estimates of kd), mutation analysis and methylation interference. Molecular weight estimates of binding protein will be obtained by UV crosslinking and/or Southwestern blotting. Since these identified sites all resemble recognition sites for AP2, mobility supershift experiments with AP2 antibodies will test for the presence of AP2 or an immunochemically related protein in the DNA/protein complexes. cDNA as encoding TGK transcription factors will be cloned either by screening an expression library with binding site probes or after partial sequence analysis of the purified protein and PCR amplification. The second part of the proposal investigates the regulation of keratinocyte transcription by arsenate, an agent shown by this lab to increase substantially cellular phosphotyrosine levels, to suppress involucrin (and to a lesser extent TGK) expression and to decrease greatly AP2 binding and AP1 transactivating activities. First, elements of the involucrin promoter required for arsenate sensitivity will be determined by transient transfections and arsenate-induced alterations in protein binding will be sought using footprinting. Second, molecular mechanisms will be investigated for suppression oa AP2 DNA binding (including alternative splicing, postranslational modification and protein/protein interactions) AP1 transactivating activity (Jun and Fos family members, phosphorylation status). Comparison of TGK and involucrin promoter elements that confer transcriptional activity may provide insight into aspects of common and distinct regulation. Of particular interest is the frequent occurrence of AP2-like elements that contribute to transcription of TGK, involucrin and other keratinocyte genes.